- Email: [email protected]
On the interactive role of central noradrenaline neurons and corticosterone in two-way active avoidance acquisition in the rat
Neuroscience Letters, 27 (1981) 341-346
341
Elsevier/North-Holland Scientific Publishers Ltd.
ON T H E I N T E R A C T I V E ROLE OF C E N T R A L N O R A D R E N A L I N E NEURONS AND CORTICOSTERONE
IN TWO-WAY ACTIVE AVOIDANCE
A C Q U I S I T I O N I N T H E RAT
T. ARCHER, S.-O. OGREN, K. FUXE*, L.F. AGNATI** and P. ENEROTH*** Research Laboratories, Astra Pharmaceuticals, SOderttilje (Sweden) *Department of Histology, Karolinska Institutet, Stockholm (Sweden) **Department of Human Physiology, University of Modena, Modena (Italy)
and ***Hormone Laboratory, Department of Obstetrics and Gynaecology, Karolinska Hospital, Stockholm (Sweden)
(Received August 27th, 1981; Revised version received October 8th, 1981; Accepted October 12th, 1981) Key words: two-way active avoidance - DSP4 - corticosterone noradrenaline - rat - pituitary-adrenal
axis - learning - noradrenaline neurotoxin
The noradrenaline (NA) neurotoxin, DSP4, caused a marked impairment of two-way active avoidance acquisition. Pretreatment with desipramine, which inhibits the degeneration of NA neurons by DSP4, consistently blocked the avoidance deficit. Daily treatment with corticosterone (2 x 1 mg/kg, s.c.) also blocked the impaired acquisition of avoidance induced by DSP4 but failed to affect the increase in cortical j3-noradrenergic receptors induced by DSP4. The present findings give further evidence for the important role interactions between the pituitary-adrenal axis and the locus coeruleus NA system play in aversive learning.
In spite o f s o m e existing evidence [2, 5 - 7 ] i m p l i c a t i n g the h y p o t h e s i z e d role o f b r a i n n o r a d r e n a l i n e ( N A ) in the acquisitive stage o f learning [5, 11], a c o n s i d e r a b l e b u l k o f evidence, m u c h o f it recent, fails to p o s t u l a t e a n y i n v o l v e m e n t for N A p a t h w a y s d u r i n g the a c q u i s i t i o n o f either negatively- o r p o s i t i v e l y - r e i n f o r c e d tasks [13, 15, 17]. T h e studies which f o u n d a role for N A in a c q u i s i t i o n utilized electrolytic lesion o f the locus coeruleus (LC) [2], whereas the use o f i n t r a c e r e b r a l injections o f the c a t e c h o l a m i n e - s e l e c t i v e n e u r o t o x i n 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) into the fibres o f the a s c e n d i n g (dorsal) N A b u n d l e (DB) failed to i m p l i c a t e N A [14, 17]. It was, h o w e v e r , f o u n d t h a t the 6 - O H D A DB lesion when c o m b i n e d with b i l a t e r a l a d r e n a l e c t o m y p r o d u c e d an i m p a i r m e n t o f r a t s ' a b i l i t y to acquire b o t h one- a n d t w o - w a y active as well as passive a v o i d a n c e learning [ 1 6 - 1 8 , 20, 21]. This i m p a i r m e n t was c o m p l e t e l y b l o c k e d b y the daily p o s t o p e r a t i v e t r e a t m e n t with 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
342 corticosterone (Cort; 2 x 1 mg/kg, s.c.), leading the authors to conclude a humoral as well as a neural involvement in the 6-OHDA ( D B ) + adrenalectomy learning deficit [18]. Recently, it was found that the new selective NA neurotoxin DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) [9, 10, 22, 23], injected systemically (50 mg/kg), caused a marked degeneration of NA neurons in several brain regions accompanied by impairments of both one- and two-way avoidance tasks [3, 19]. Since dopamine (DA) was left untouched, and the acquisition deficits as well as the degeneration of NA neurons were reliably blocked by the selective NA uptake inhibitor desipramine (DMI), it was concluded that the DSP4-induced destruction of the NA-LC system in its entire extent was responsible for the disruption of aversive learning. In view of the findings that Cort attenuated the disruptive effects of 6-OHDA DB lesions when combined with bilateral adrenalectomy, it is possible that systemic DSP4 may to some extent mimic the action of 6-OHDA + adrenalectomy. If this was the case then the post-DSP4 administration of daily corticosterone treatment might result in either a complete or partial blockade of the observed avoidance acquisition impairment. Thus, two experiments were designed to study: (a) the effects of daily Cort treatment on the two-way active avoidance deficit induced by DSP4, and (b) the ability of Cort to block the supersensitivity development at cortical ~-adrenergic receptors induced by the DSP4-induced degeneration of the cortical NA networks. Male Sprague-Dawley rats, weighing 250-285 g (aged 55-60 days), and allowed at least 10 days adaption to the laboratory conditions, were used in both experiments. In Experiment I, 4 grups (n = 8) were used and half of the total of 32 rats received intraperitoneal (i.p.) injections of DSP4 (50 mg/kg, dissolved in distilled water) while the other half received i.p. injections of distilled water (5 ml/kg) 7 days prior to the onset of the first avoidance session. Eight DSP4 and 8 distilled water (control) rats received daily subcutaneous (s.c.) injections of Cort (2 x 1 mg/kg, dissolved in 5% ethanol), the first of which was administered 2-3 h following DSP4 at 08.00 h and the second at about 16.00 h. The injections continued during the whole experiment. The remaining 8 DSP4 and 8 control rats received daily injections (s.c.) of saline (1 mg/kg) up to the time of and during the experiment. In Experiment II, 4 groups were again used and 32 rats were injected (i.p.) with either DSP4 (50 mg/kg, n = 16), DMI (20 mg/kg, 30 min prior to DSP4) + DSP4 (50 mg/kg, n = 8), or distilled water (5 ml/kg, n = 8). Half the DSP4 rats received daily Cort injections (2 x 1 mg/kg, s.c., n = 8) using the same procedure as in Experiment I. In a separate experiment 4 groups of rats (n = 8) were given identical treatments to those given in Experiment II: DSP4 (50 mg/kg), DMI (20 mg/kg) + DSP4, DSP4 (50 mg/kg) + daily Cort (2 x 1 mg/kg, s.c.) and distilled water (5 ml/kg). The rats were killed by decapitation on the 10th day after DSP4 treatment (4-5 h following the last Cort injection). The cortex cerebri, located dorsally to a horizontal plane passing immediately above the dorsal surface of the
343 nuc. caudatus, was dissected out on ice and stored at - 7 0 ° C until analysis of /3-adrenergic receptors, using the radioligand [3H]-dihydroalprenolol ([3H]DHA) (44.9 Ci/mmol; NEN, Boston), a 13-adrenergic antagonist. The binding procedure was that of Bylund and Snyder [4]. The cerebral cortex from the respective groups was homogenized in a 0.05 M Tris-HCl buffer (pH = 8.0 at 25 °C) using a sonifiercell disruptor (B-30, setting 7, 60 sec). The [3H]DHA binding assay was performed on the crude membrane fraction obtained by centrifugation twice at 50,000 × g [4]. The membrane concentration was 10 mg/ml and the filtration procedure was performed by means of a semiautomatic cell culture harvester [8] using a glass fibre paper (Whatman GF/B). The sedimented material was resuspended in 50 mM TrisHCI buffer (pH 8.0) was incubated for 30 min at room temperature. Unspecific binding was defined as the binding in the presence of 10 -6 M of (_+)-propranolol. Saturation analysis was performed to evaluate the effects of the treatments on the binding characteristics of [3H]DHA binding. Standard parametric procedures were used to fit the best straight line [24]. Daily Cort treatment (2 × 1 mg/kg) alleviated the impairment of acquisition of two-way avoidance, consistently observed in DSP4-treated rats in both Experiments I and lI. The extent to which Cort treatment attenuated the avoidance deficit induced by DSP4 was found to be less than complete in comparison with the DMI + DSP4 treated rats. Fig. 1 presents the mean per cent conditioned avoidance responses (o7o CARs) of the various groups in both experiments. The groups x sessions interaction (Split-plot ANOVA) [12] was significant in both Experiment I (F(12, 112) = 3.05, P < 0.01) and Experiment II (F(12, 112) = 2.49, P < 0.01). Scheff~'s non-pairwise test (12), used to compare the DSP4 group with the DSP4 + Cort, Cort and control groups (see Fig. 1A), indicated significantly fewer CARs by the DSP4 rats during sessions 2-5. No differences in the number of intertrial crossings (ITCs) were observed. Cort treatment by itself did not alter the acquisition of two-way avoidance. In Experiment II, the DSP4 group made significantly fewer CARs than the DSP4 + Cort group during sessions 4 and 5 (t-test, P < 0.02), which in turn performed significantly fewer CARs than the DMI + DSP4 and control groups (Scheff~'s test, P < 0.01) during sessions 2-5. Although the groups × sessions interaction for the ITC data was significant (F(12, 112) = 2.31, P < 0.01), the only significant effect noted was significantly fewer numbers of ITCS made by the DSP4 + Cort group compared with the DSP4 and control groups (t-test, P < 0.05) during session 5. Degeneration of cortical NA nerve terminal by DSP4 resulted in a marked and a significant increase in the number of [3H]DHA binding sites without any significant changes in K o values (Fig. 2). DMI pretreatment significantly attenuated the increase of [3H]DHA binding sites while Cort treatment failed to affect this increase (Fig. 2). Systemically injected DSP4 (50 mg/kg, i.p.), like the 6-OHDA dorsal NA bundle lesion + adrenalectomy combination [20], caused a disruption of rats' ability to
344
'~0 CARs 100 F
Cot t DSP4 • cort Control DS P4
60 i
% CARs 100 80
Cort
+o
++/.%:
_
,o
20!
Control OMI'DSP4 DSP4"corI DSP4
~ ' ~ ' D S P 4
OSP4. cort ~
"DSP4
20
0L
0
1
i
3
4
;
1
SESSION NUMBER
2 3 4 5 SESSION NUMBER
Fig. 1. The mean per cent conditioned avoidance responses (% CARs) during each of 5 consecutive twoway active avoidance sessions for the various groups in Experiments 1 and II (for treatments, see text). Groups embraced by the same line in the tables of the figure are not significantly different. A Scheff+'s test indicated a significant difference at the 1070 level when comparing the Cort, DSP4 + Cort and control groups with the DSP4 groups during sessions 2-5 (right). Scheffb's test indicated a significant difference at the 1% level when comparing the DMI + DSP4 and control groups with the DSP4 + Cort group during sessions 2-5; t-test indicated a significant difference between the DSP4 + Cort and DSP4 groups at the 2% level during the final two sessions (see text). Two-way active avoidance acquisition took place as described previously [19]. Each trial consisted of a 10 sec tone (1000 Hz) presentation followed by a 5 sec shock (1.0 mA). A 40 sec intertrial interval was maintained and 30 trials were presented during each daily session.
Control KD-O,SlnM Bmx=4.82 pmol/g i
20 15
DMI+DSP4 KD=O.37nM Bmx=5.33 pmol/g
0
C
043nMDSP4
•
KD=0.3 nM
\ 1
5
10 1
5
10
(pmol/9~
Fig. 2. The effects of DMI (20 mg/k, 30 rain prior to DSP4) and corticosterone (daily, 2 x 1 mg/kg, s.c.) on the DSP4-induced increase of cortical/%adrenergic receptors as evaluated on [3H]DHA binding (for details, see text). Saturation analysis was performed and the Scatchard plot is shown. The concentration of [3H]DHA ranged from 0.1 to 5 nM. Each point represents the mean of 4 values. The experiment has been repeated twice.
345
acquire the two-way active avoidance task in both Experiments I and II. Within the time-span of this experiment the peripheral NA neurons are only marginally affected, showing a 25% reduction of NA stores at 7 days. This effect has disappeared completely after 14 days [3]. Furthermore, 6-OHDA given systemically produces a marked degeneration of peripheral NA neurons and in spite of this action fails to affect two-way active avoidance acquisition [3]. The DSP4-induced deficit in the acquisition of two-way avoidance, again like the 6-OHDA DB ÷ adrenalectomy combination, was alleviated by daily Cort treatment. Several explanations for the attenuation of the DSP4 deficit by Cort may be considered. There is no evidence that Cort might block the NA degeneration induced by DSP4 via a presynaptic mechanism, e.g. analogous to the blocking action of DMI. Alternatively, Cort treatment may alter postsynaptic NA mechanisms which may be essential. DSP4 treatment leads to a pronounced increase in fl-receptor binding after 7 (Fig. 2) and 14 days [10]. Prior DMI administration, which blocks degeneration of the NA nerve terminals, inhibited the increase in fl-adrenergic receptors induced by the systemic administration of DSP4. Daily Cort treatment, on the other hand, did not. This contrasts with the effects of adrenalectomy which effectively counteracts the increase in [3H]DHA binding caused by 6-OHDA lesions of the dorsal bundle [20]. These findings seem to exclude the possibility that Cort may have influenced the adaptive changes in fl-adrenergic recognition sites in the dorsal cortex seen following the loss of NA terminals, although such an influence of Cort may exist in other brain regions. However, it is possible that Cort can modulate transmission at fl-adrenergic receptor sites via effects on adenylate cyclase and/or on coupling proteins (see refs. 1 and 20). Obviously, Cort can also act via other types of central synapses. It must be emphasized that the action of Cort are not induced via alleviation of shock-induced suppression of locomotor activity (stress related disruption), since avoidance performance has been shown in these rats to be independent of locomotor functions [3]. The DSP4 + Cort group did not differ from the control group in the first experiment but did so in the second; however, the DSP4 + Cort group did differ from the DSP4 group in both cases. Daily cort treatment may only partially alleviate the avoidance deficit caused by DSP4. However, both sets of data underline the interactive role of the locus coeruleus NA system and the pituitary-adrenal axis in aversive learning processes, possibly by their participation in the same local circuits of the tel- and diencephalon. The excellent technical assistence rendered by G0ran Johansson is gratefully acknowledged. This work has, in part, been supported by a grant (O4X-715) from the Swedish Medical Research Council. 1 Agnati, L.F., Fuxe, K., Kuonen, D., Blake, C.A., Andersson, K., Eneroth, P., Gustafsson, J.-,~., Battistini, N. and Calza, L., Effects of estrogen and progesterone on central c~- and B-adrenergic receptors in ovariectomized rats: evidence for gonadal steroid receptor regulation of brain a- and
346
2 3
4 5 6 7 8 9 10
11 12
/3-adrenergic receptors. In K. Fuxe, j._~i,. Gustafsson and L. Wetterberg (Eds.), Steroid Hormone Regulation of the Brain, Pergamon Press, Oxford, 1981, pp. 237 252. Anlezark, G.M., Crow, T.J. and Greenway, A.P., Impaired learning and decreased cortical norepinephrine after bilateral locus coeruleus lesions, Science, 181 (1973) 682-684. Archer, T., Ogren, S.-O., Ross, S.B. and Johansson, G., Two-way active avoidance, intertrial crossings and locomotor activity following systemic administration of DSP4, a new noradrenaline neurotoxin, in the rat, Psychopharmacology, submitted. Bylund, D.B. and Snyder, S.H., Beta adrenergic receptor binding in membrane preparations from mammalian brain, Molec. Pharmacol., 12 (1976) 568-580. Crow, T.J., The coerulo-cortical norepinephrine system and learning, In E. Usdin and S.H. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, New York, 1973, pp. 723-726. Crow, T.J., Spear, P.J. and Arbuthnott, G.W., lntracranial selfstimulation with electrodes in the region of the locus coeruleus, Brain Res., 36 (1972) 275- 287. Fuxe, K. and Hansson, I,.C.F., Central catecholamine neurones and conditioned avoidance behaviour, Psychopharmacology, I1 (1967)439-447. Hall, H. and Thor, L., Evaluation of a semiautomatic filtration technique receptor binding studies, Life Sci., 24 (1979) 2393-2400. Jaim-Etcheverry, G. and Zieher, L.M., DSP4: a novel compound with neurotoxic effect on noradrenaline neurons of adult and developing rats, Brain Res., 188 (1980) 513 523. Jonsson, G., Hallman, H., Ponzio, F. and Ross, S.B. DSP4 - (N-2-chloroethyl-N-ethyl-2-bromobenzylamine) a useful denervation tool for central and peripheral noradrenaline neurons, Europ. J. Pharmacol., in press. Kety, S.S., The possible role of the adrenergic system of the cortex in learning, Res. Publ. Ass. Res. nerv. Ment. Dis., 50 (1970) 276 289. Kirk, R.E., Experimental Design: Procedures for the behavioural sciences, Brooks/Cole, Belmont,
1968. 13 Mason, S.T. and Fibiger, H.C., Noradrenaline and avoidance learning in the rat, Brain Res., 161 (1979) 321-334. 14 Mason, S.T. and lversen, S.D., Learning in the absence of forebrain noradrenaline, Nature (Lond.), 258 (1975) 422-424. 15 Mason, S.T. and lversen, S.D., Theories of the dorsal bundle extinction effect, Brain Res. Rev., 1 (1979) 107-137. 16 Mason, S.T., Roberts, D.C.S. and Fibiger, H.C., Interaction of brain noradrenaline and the pituitary adrenal axis in learning and extinction, Pharmacol. Biochem. Bebav., 10 (1978) 11-16. 17 Ogren, S.-O. and Fuxe, K., Learning, brain noradrenaline and the pituitary-adrenal axis, Med. Biol., 52 (1974) 399-405. 18 Ogren, S.-O. and Fuxe, K., On the role of noradrenaline and the pituitary-adrenal axis in avoidance learning. Studies with corticosterone, Neurosci. Lett., 5 (1977) 291-296. 19 Ogren, S.-O., Archer, T. and Ross, S.B,, Evidence for a role of the locus coeruleus noradrenaline system in learning, Neurosci. Lett., 20 (1980) 351-356. 20 t)gren, S.-O., Archer, T., Fuxe, K. Eneroth, P. and Agnati, L.F., Glucocorticoids, catecholamines and avoidance learning. In. K. Fuxe, J.-,k. Gustafsson and I_. Wetterberg (Eds.), Steroid Hormone Regulation of the Brain, Pergamon Press, 1981, pp. 355 377. 21 Roberts, D.C.S. and Fibiger, H.C., Evidence for interactions between central noradrenaline neurons and adrenal hormones in learning and memory, Pbarmacol. Biochem. Behav., 7 (1977) 191-194. 22 Ross, S.B., Long-term effects of N-2-chloroethyl-N-ethyl-2-bromobenzylamine hydrochloride on noradrenergic neurones in the rat brain and heart, Br. J. Pharmacol., 58 (1976) 524-527. 23 Ross, S.B. and Renyi, A.L., On the long-lasting inhibition effects of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) on the active uptake of noradrenaline, J. Pharm. Pharmacol., 28 (1976) 458 459. 24 Snedecor, G.W. and Cochran, W.G., Statistical Methods, Iowa State University Press, Ames, 1972.
341
Elsevier/North-Holland Scientific Publishers Ltd.
ON T H E I N T E R A C T I V E ROLE OF C E N T R A L N O R A D R E N A L I N E NEURONS AND CORTICOSTERONE
IN TWO-WAY ACTIVE AVOIDANCE
A C Q U I S I T I O N I N T H E RAT
T. ARCHER, S.-O. OGREN, K. FUXE*, L.F. AGNATI** and P. ENEROTH*** Research Laboratories, Astra Pharmaceuticals, SOderttilje (Sweden) *Department of Histology, Karolinska Institutet, Stockholm (Sweden) **Department of Human Physiology, University of Modena, Modena (Italy)
and ***Hormone Laboratory, Department of Obstetrics and Gynaecology, Karolinska Hospital, Stockholm (Sweden)
(Received August 27th, 1981; Revised version received October 8th, 1981; Accepted October 12th, 1981) Key words: two-way active avoidance - DSP4 - corticosterone noradrenaline - rat - pituitary-adrenal
axis - learning - noradrenaline neurotoxin
The noradrenaline (NA) neurotoxin, DSP4, caused a marked impairment of two-way active avoidance acquisition. Pretreatment with desipramine, which inhibits the degeneration of NA neurons by DSP4, consistently blocked the avoidance deficit. Daily treatment with corticosterone (2 x 1 mg/kg, s.c.) also blocked the impaired acquisition of avoidance induced by DSP4 but failed to affect the increase in cortical j3-noradrenergic receptors induced by DSP4. The present findings give further evidence for the important role interactions between the pituitary-adrenal axis and the locus coeruleus NA system play in aversive learning.
In spite o f s o m e existing evidence [2, 5 - 7 ] i m p l i c a t i n g the h y p o t h e s i z e d role o f b r a i n n o r a d r e n a l i n e ( N A ) in the acquisitive stage o f learning [5, 11], a c o n s i d e r a b l e b u l k o f evidence, m u c h o f it recent, fails to p o s t u l a t e a n y i n v o l v e m e n t for N A p a t h w a y s d u r i n g the a c q u i s i t i o n o f either negatively- o r p o s i t i v e l y - r e i n f o r c e d tasks [13, 15, 17]. T h e studies which f o u n d a role for N A in a c q u i s i t i o n utilized electrolytic lesion o f the locus coeruleus (LC) [2], whereas the use o f i n t r a c e r e b r a l injections o f the c a t e c h o l a m i n e - s e l e c t i v e n e u r o t o x i n 6 - h y d r o x y d o p a m i n e ( 6 - O H D A ) into the fibres o f the a s c e n d i n g (dorsal) N A b u n d l e (DB) failed to i m p l i c a t e N A [14, 17]. It was, h o w e v e r , f o u n d t h a t the 6 - O H D A DB lesion when c o m b i n e d with b i l a t e r a l a d r e n a l e c t o m y p r o d u c e d an i m p a i r m e n t o f r a t s ' a b i l i t y to acquire b o t h one- a n d t w o - w a y active as well as passive a v o i d a n c e learning [ 1 6 - 1 8 , 20, 21]. This i m p a i r m e n t was c o m p l e t e l y b l o c k e d b y the daily p o s t o p e r a t i v e t r e a t m e n t with 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
342 corticosterone (Cort; 2 x 1 mg/kg, s.c.), leading the authors to conclude a humoral as well as a neural involvement in the 6-OHDA ( D B ) + adrenalectomy learning deficit [18]. Recently, it was found that the new selective NA neurotoxin DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) [9, 10, 22, 23], injected systemically (50 mg/kg), caused a marked degeneration of NA neurons in several brain regions accompanied by impairments of both one- and two-way avoidance tasks [3, 19]. Since dopamine (DA) was left untouched, and the acquisition deficits as well as the degeneration of NA neurons were reliably blocked by the selective NA uptake inhibitor desipramine (DMI), it was concluded that the DSP4-induced destruction of the NA-LC system in its entire extent was responsible for the disruption of aversive learning. In view of the findings that Cort attenuated the disruptive effects of 6-OHDA DB lesions when combined with bilateral adrenalectomy, it is possible that systemic DSP4 may to some extent mimic the action of 6-OHDA + adrenalectomy. If this was the case then the post-DSP4 administration of daily corticosterone treatment might result in either a complete or partial blockade of the observed avoidance acquisition impairment. Thus, two experiments were designed to study: (a) the effects of daily Cort treatment on the two-way active avoidance deficit induced by DSP4, and (b) the ability of Cort to block the supersensitivity development at cortical ~-adrenergic receptors induced by the DSP4-induced degeneration of the cortical NA networks. Male Sprague-Dawley rats, weighing 250-285 g (aged 55-60 days), and allowed at least 10 days adaption to the laboratory conditions, were used in both experiments. In Experiment I, 4 grups (n = 8) were used and half of the total of 32 rats received intraperitoneal (i.p.) injections of DSP4 (50 mg/kg, dissolved in distilled water) while the other half received i.p. injections of distilled water (5 ml/kg) 7 days prior to the onset of the first avoidance session. Eight DSP4 and 8 distilled water (control) rats received daily subcutaneous (s.c.) injections of Cort (2 x 1 mg/kg, dissolved in 5% ethanol), the first of which was administered 2-3 h following DSP4 at 08.00 h and the second at about 16.00 h. The injections continued during the whole experiment. The remaining 8 DSP4 and 8 control rats received daily injections (s.c.) of saline (1 mg/kg) up to the time of and during the experiment. In Experiment II, 4 groups were again used and 32 rats were injected (i.p.) with either DSP4 (50 mg/kg, n = 16), DMI (20 mg/kg, 30 min prior to DSP4) + DSP4 (50 mg/kg, n = 8), or distilled water (5 ml/kg, n = 8). Half the DSP4 rats received daily Cort injections (2 x 1 mg/kg, s.c., n = 8) using the same procedure as in Experiment I. In a separate experiment 4 groups of rats (n = 8) were given identical treatments to those given in Experiment II: DSP4 (50 mg/kg), DMI (20 mg/kg) + DSP4, DSP4 (50 mg/kg) + daily Cort (2 x 1 mg/kg, s.c.) and distilled water (5 ml/kg). The rats were killed by decapitation on the 10th day after DSP4 treatment (4-5 h following the last Cort injection). The cortex cerebri, located dorsally to a horizontal plane passing immediately above the dorsal surface of the
343 nuc. caudatus, was dissected out on ice and stored at - 7 0 ° C until analysis of /3-adrenergic receptors, using the radioligand [3H]-dihydroalprenolol ([3H]DHA) (44.9 Ci/mmol; NEN, Boston), a 13-adrenergic antagonist. The binding procedure was that of Bylund and Snyder [4]. The cerebral cortex from the respective groups was homogenized in a 0.05 M Tris-HCl buffer (pH = 8.0 at 25 °C) using a sonifiercell disruptor (B-30, setting 7, 60 sec). The [3H]DHA binding assay was performed on the crude membrane fraction obtained by centrifugation twice at 50,000 × g [4]. The membrane concentration was 10 mg/ml and the filtration procedure was performed by means of a semiautomatic cell culture harvester [8] using a glass fibre paper (Whatman GF/B). The sedimented material was resuspended in 50 mM TrisHCI buffer (pH 8.0) was incubated for 30 min at room temperature. Unspecific binding was defined as the binding in the presence of 10 -6 M of (_+)-propranolol. Saturation analysis was performed to evaluate the effects of the treatments on the binding characteristics of [3H]DHA binding. Standard parametric procedures were used to fit the best straight line [24]. Daily Cort treatment (2 × 1 mg/kg) alleviated the impairment of acquisition of two-way avoidance, consistently observed in DSP4-treated rats in both Experiments I and lI. The extent to which Cort treatment attenuated the avoidance deficit induced by DSP4 was found to be less than complete in comparison with the DMI + DSP4 treated rats. Fig. 1 presents the mean per cent conditioned avoidance responses (o7o CARs) of the various groups in both experiments. The groups x sessions interaction (Split-plot ANOVA) [12] was significant in both Experiment I (F(12, 112) = 3.05, P < 0.01) and Experiment II (F(12, 112) = 2.49, P < 0.01). Scheff~'s non-pairwise test (12), used to compare the DSP4 group with the DSP4 + Cort, Cort and control groups (see Fig. 1A), indicated significantly fewer CARs by the DSP4 rats during sessions 2-5. No differences in the number of intertrial crossings (ITCs) were observed. Cort treatment by itself did not alter the acquisition of two-way avoidance. In Experiment II, the DSP4 group made significantly fewer CARs than the DSP4 + Cort group during sessions 4 and 5 (t-test, P < 0.02), which in turn performed significantly fewer CARs than the DMI + DSP4 and control groups (Scheff~'s test, P < 0.01) during sessions 2-5. Although the groups × sessions interaction for the ITC data was significant (F(12, 112) = 2.31, P < 0.01), the only significant effect noted was significantly fewer numbers of ITCS made by the DSP4 + Cort group compared with the DSP4 and control groups (t-test, P < 0.05) during session 5. Degeneration of cortical NA nerve terminal by DSP4 resulted in a marked and a significant increase in the number of [3H]DHA binding sites without any significant changes in K o values (Fig. 2). DMI pretreatment significantly attenuated the increase of [3H]DHA binding sites while Cort treatment failed to affect this increase (Fig. 2). Systemically injected DSP4 (50 mg/kg, i.p.), like the 6-OHDA dorsal NA bundle lesion + adrenalectomy combination [20], caused a disruption of rats' ability to
344
'~0 CARs 100 F
Cot t DSP4 • cort Control DS P4
60 i
% CARs 100 80
Cort
+o
++/.%:
_
,o
20!
Control OMI'DSP4 DSP4"corI DSP4
~ ' ~ ' D S P 4
OSP4. cort ~
"DSP4
20
0L
0
1
i
3
4
;
1
SESSION NUMBER
2 3 4 5 SESSION NUMBER
Fig. 1. The mean per cent conditioned avoidance responses (% CARs) during each of 5 consecutive twoway active avoidance sessions for the various groups in Experiments 1 and II (for treatments, see text). Groups embraced by the same line in the tables of the figure are not significantly different. A Scheff+'s test indicated a significant difference at the 1070 level when comparing the Cort, DSP4 + Cort and control groups with the DSP4 groups during sessions 2-5 (right). Scheffb's test indicated a significant difference at the 1% level when comparing the DMI + DSP4 and control groups with the DSP4 + Cort group during sessions 2-5; t-test indicated a significant difference between the DSP4 + Cort and DSP4 groups at the 2% level during the final two sessions (see text). Two-way active avoidance acquisition took place as described previously [19]. Each trial consisted of a 10 sec tone (1000 Hz) presentation followed by a 5 sec shock (1.0 mA). A 40 sec intertrial interval was maintained and 30 trials were presented during each daily session.
Control KD-O,SlnM Bmx=4.82 pmol/g i
20 15
DMI+DSP4 KD=O.37nM Bmx=5.33 pmol/g
0
C
043nMDSP4
•
KD=0.3 nM
\ 1
5
10 1
5
10
(pmol/9~
Fig. 2. The effects of DMI (20 mg/k, 30 rain prior to DSP4) and corticosterone (daily, 2 x 1 mg/kg, s.c.) on the DSP4-induced increase of cortical/%adrenergic receptors as evaluated on [3H]DHA binding (for details, see text). Saturation analysis was performed and the Scatchard plot is shown. The concentration of [3H]DHA ranged from 0.1 to 5 nM. Each point represents the mean of 4 values. The experiment has been repeated twice.
345
acquire the two-way active avoidance task in both Experiments I and II. Within the time-span of this experiment the peripheral NA neurons are only marginally affected, showing a 25% reduction of NA stores at 7 days. This effect has disappeared completely after 14 days [3]. Furthermore, 6-OHDA given systemically produces a marked degeneration of peripheral NA neurons and in spite of this action fails to affect two-way active avoidance acquisition [3]. The DSP4-induced deficit in the acquisition of two-way avoidance, again like the 6-OHDA DB ÷ adrenalectomy combination, was alleviated by daily Cort treatment. Several explanations for the attenuation of the DSP4 deficit by Cort may be considered. There is no evidence that Cort might block the NA degeneration induced by DSP4 via a presynaptic mechanism, e.g. analogous to the blocking action of DMI. Alternatively, Cort treatment may alter postsynaptic NA mechanisms which may be essential. DSP4 treatment leads to a pronounced increase in fl-receptor binding after 7 (Fig. 2) and 14 days [10]. Prior DMI administration, which blocks degeneration of the NA nerve terminals, inhibited the increase in fl-adrenergic receptors induced by the systemic administration of DSP4. Daily Cort treatment, on the other hand, did not. This contrasts with the effects of adrenalectomy which effectively counteracts the increase in [3H]DHA binding caused by 6-OHDA lesions of the dorsal bundle [20]. These findings seem to exclude the possibility that Cort may have influenced the adaptive changes in fl-adrenergic recognition sites in the dorsal cortex seen following the loss of NA terminals, although such an influence of Cort may exist in other brain regions. However, it is possible that Cort can modulate transmission at fl-adrenergic receptor sites via effects on adenylate cyclase and/or on coupling proteins (see refs. 1 and 20). Obviously, Cort can also act via other types of central synapses. It must be emphasized that the action of Cort are not induced via alleviation of shock-induced suppression of locomotor activity (stress related disruption), since avoidance performance has been shown in these rats to be independent of locomotor functions [3]. The DSP4 + Cort group did not differ from the control group in the first experiment but did so in the second; however, the DSP4 + Cort group did differ from the DSP4 group in both cases. Daily cort treatment may only partially alleviate the avoidance deficit caused by DSP4. However, both sets of data underline the interactive role of the locus coeruleus NA system and the pituitary-adrenal axis in aversive learning processes, possibly by their participation in the same local circuits of the tel- and diencephalon. The excellent technical assistence rendered by G0ran Johansson is gratefully acknowledged. This work has, in part, been supported by a grant (O4X-715) from the Swedish Medical Research Council. 1 Agnati, L.F., Fuxe, K., Kuonen, D., Blake, C.A., Andersson, K., Eneroth, P., Gustafsson, J.-,~., Battistini, N. and Calza, L., Effects of estrogen and progesterone on central c~- and B-adrenergic receptors in ovariectomized rats: evidence for gonadal steroid receptor regulation of brain a- and
346
2 3
4 5 6 7 8 9 10
11 12
/3-adrenergic receptors. In K. Fuxe, j._~i,. Gustafsson and L. Wetterberg (Eds.), Steroid Hormone Regulation of the Brain, Pergamon Press, Oxford, 1981, pp. 237 252. Anlezark, G.M., Crow, T.J. and Greenway, A.P., Impaired learning and decreased cortical norepinephrine after bilateral locus coeruleus lesions, Science, 181 (1973) 682-684. Archer, T., Ogren, S.-O., Ross, S.B. and Johansson, G., Two-way active avoidance, intertrial crossings and locomotor activity following systemic administration of DSP4, a new noradrenaline neurotoxin, in the rat, Psychopharmacology, submitted. Bylund, D.B. and Snyder, S.H., Beta adrenergic receptor binding in membrane preparations from mammalian brain, Molec. Pharmacol., 12 (1976) 568-580. Crow, T.J., The coerulo-cortical norepinephrine system and learning, In E. Usdin and S.H. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, New York, 1973, pp. 723-726. Crow, T.J., Spear, P.J. and Arbuthnott, G.W., lntracranial selfstimulation with electrodes in the region of the locus coeruleus, Brain Res., 36 (1972) 275- 287. Fuxe, K. and Hansson, I,.C.F., Central catecholamine neurones and conditioned avoidance behaviour, Psychopharmacology, I1 (1967)439-447. Hall, H. and Thor, L., Evaluation of a semiautomatic filtration technique receptor binding studies, Life Sci., 24 (1979) 2393-2400. Jaim-Etcheverry, G. and Zieher, L.M., DSP4: a novel compound with neurotoxic effect on noradrenaline neurons of adult and developing rats, Brain Res., 188 (1980) 513 523. Jonsson, G., Hallman, H., Ponzio, F. and Ross, S.B. DSP4 - (N-2-chloroethyl-N-ethyl-2-bromobenzylamine) a useful denervation tool for central and peripheral noradrenaline neurons, Europ. J. Pharmacol., in press. Kety, S.S., The possible role of the adrenergic system of the cortex in learning, Res. Publ. Ass. Res. nerv. Ment. Dis., 50 (1970) 276 289. Kirk, R.E., Experimental Design: Procedures for the behavioural sciences, Brooks/Cole, Belmont,
1968. 13 Mason, S.T. and Fibiger, H.C., Noradrenaline and avoidance learning in the rat, Brain Res., 161 (1979) 321-334. 14 Mason, S.T. and lversen, S.D., Learning in the absence of forebrain noradrenaline, Nature (Lond.), 258 (1975) 422-424. 15 Mason, S.T. and lversen, S.D., Theories of the dorsal bundle extinction effect, Brain Res. Rev., 1 (1979) 107-137. 16 Mason, S.T., Roberts, D.C.S. and Fibiger, H.C., Interaction of brain noradrenaline and the pituitary adrenal axis in learning and extinction, Pharmacol. Biochem. Bebav., 10 (1978) 11-16. 17 Ogren, S.-O. and Fuxe, K., Learning, brain noradrenaline and the pituitary-adrenal axis, Med. Biol., 52 (1974) 399-405. 18 Ogren, S.-O. and Fuxe, K., On the role of noradrenaline and the pituitary-adrenal axis in avoidance learning. Studies with corticosterone, Neurosci. Lett., 5 (1977) 291-296. 19 Ogren, S.-O., Archer, T. and Ross, S.B,, Evidence for a role of the locus coeruleus noradrenaline system in learning, Neurosci. Lett., 20 (1980) 351-356. 20 t)gren, S.-O., Archer, T., Fuxe, K. Eneroth, P. and Agnati, L.F., Glucocorticoids, catecholamines and avoidance learning. In. K. Fuxe, J.-,k. Gustafsson and I_. Wetterberg (Eds.), Steroid Hormone Regulation of the Brain, Pergamon Press, 1981, pp. 355 377. 21 Roberts, D.C.S. and Fibiger, H.C., Evidence for interactions between central noradrenaline neurons and adrenal hormones in learning and memory, Pbarmacol. Biochem. Behav., 7 (1977) 191-194. 22 Ross, S.B., Long-term effects of N-2-chloroethyl-N-ethyl-2-bromobenzylamine hydrochloride on noradrenergic neurones in the rat brain and heart, Br. J. Pharmacol., 58 (1976) 524-527. 23 Ross, S.B. and Renyi, A.L., On the long-lasting inhibition effects of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) on the active uptake of noradrenaline, J. Pharm. Pharmacol., 28 (1976) 458 459. 24 Snedecor, G.W. and Cochran, W.G., Statistical Methods, Iowa State University Press, Ames, 1972.